The goal is to establish the exact role of guanine nucleotide in the cholera toxin activation of adenylate cyclase. By simplifying the conditions for toxin treatment of plasma membranes and by using highly purified ATP as a substrate for enzyme assay, we found that the expression of activity for toxin activitated adenylate cyclase has an absolute requirement for GTP. These results were interpreted by assuming that the component responsible for GTP activation is a specific GTPase and that cholera toxin treatment leads to inhibition of this GTPase, allowing GTP to activate adenylate cyclase in a manner similar to GppNHp and GpCH2pp, analogs resistant to GTPase hydrolysis. This research suggests that modulation of adenylate cyclase through the GTP component may be an important mechanism for intracellular regulation of cyclic AMP levels.